Optimized Synthesis Of Pure, Non-Polymorphic, Crystalline Bile Acids With Defined Particle Size

ABSTRACT

The present invention relates to a pure polymorph of Nor-UDCA or Bis-nor-UDCA, or of a pharmaceutically acceptable salt thereof. The inventions further provides a pharmaceutical composition comprising the polymorph of the invention, and a method for preparing the polymorph. The invention includes the pharmaceutical use of the polymorph or of the pharmaceutical composition of the invention.

The present invention is related to a pharmaceutical, production suitedsynthesis exceptionally applicable for the treatment of bile- and liverdiseases, which contains norursodeoxycholic acid (Nor-UDCA),bis-nor-ursodeoxyehoiic acid (Bis-Nor-UDCA) of a pharmaceuticalacceptable salt or a derivative thereof.

BACKGROUND OF THE INVENTION

Nor-UDCA and Bis-Nor-UDCA are ursodeoxycholic acid analogs with modifiedphysiochemical properties, like solubility, critically micellarconcentration, or hydrophilicity (Roda et al., Dig Dis and Sciences,1989). A method for the synthesis of 24-nor-5βcholan-23-oic acid wasalready described by Schteingart and Hofmann (Journal of Lipid Research,1988). In vitro experiments demonstrated their efficacy in animal modelsof cholestatic liver disease (PCT/EP2005/052178). A method for thepreparation of Nor-UDCA is described in EP 0624595 B1. However, thisdocument is silent with respect to characteristic chemical and physicalproperties, purity, extent of crystallization and the particle size ofthe synthesized Nor-UDCA.

The application of bile acids, especially ursodeoxycholic acid, in thetreatment of cholestatic liver diseases, like Primary Biliary Cirrhosis,is well known and published already in the eighties of the last century(Poupon et al., Lancet, 1987). While the use of current availablepharmaceutical preparations only results in the successful treatment ofa subset of patients, there is a need for patients who do not respondthe ursodeoxycholic acid therapy or suffer from cholestatic liverdiseases or metabolic diseases that are not treatable withursodeoxycholic acid.

Depending on the pH-value of the solvent bile acids are of lowsolubility. An adaequatsty good solubility of bile acids in theintestinal tract is a prerequisite for a successful pharmaceuticaltreatment. Solubility may be improved by salt formation of Nor-UDCA. Asecond objective is a preparation with a sufficient oralbioavailability. A high in vitro-dissolution rate is a prerequisite forsufficient oral bioavailability. Micronization, e.g. the production of apharmaceutical preparation with a very small defined particle size (>60%with a diameter of less than 10 μm), is an established method toincrease the dissolution rate. A known, but elaborate process to achievemicronized particles is by extensive milling.

An additional objective of the present invention was to provide aphysically pure, e.g. crystalline, preparation, which isthermodynamically stable.

The objective of this invention was to synthesize a novel form ofNor-UDCA or Bis-Nor-UDCA at high quality, which has favourable purity,particle size characteristics and that is applicable for the treatmentof cholestatic or metabolic liver diseases. The desired crystal formshould be obtained in a consistent and reproducible manner by a scalableand industrial production process.

Since crystal modifications of a substance represent different crystalstructures with potentially different properties, the main objective ofthe invention was to identify and select the thermodynamically stablepolymorph/single crystalline form of Nor-UDCA that does not convert intoanother polymorphic form. This particular modification of Nor-UDCAshould exhibit considerable chemical and physical advantages overmetastable forms and should therefore be the substance of choice forfurther chemical and pharmaceutical development.

In addition, it is desirable to produce Nor-UDCA with a consistentparticle size and morphology because the crystal habit affects importantprocessing parameters such as flowability, bulk density andcompressibility. Micronization of Nor-UDCA is preferred to increase thedissolution rate of the compound and by this the oral bioavailability.

The conditions of the purification and crystallisation process shouldproduce the appropriate solid form of Nor-UDCA with reliable andreproducible polymorphic purity, chemical purify, crystal habit andyield. Micronization by milling in order to control the crystal size ofNor-UDCA can be avoided. Thereby, a common phenomenon uponmicronization, namely amorphization, can be prevented.

The published method for the synthesis of Nor-UDCA is not suitable tomeet pharmaceutical requirements. Especially the purification route isnot effective to reach the desired product qualities with regard topolymorphic purify, chemical purity, crystal habit and yield.Conventional methods of purification do not allow to obtain a polymorphof Nor-UDCA or Bis-nor-UDCA having a very high chemical purify, e.g.such that the total amount of impurities is less than 0.05%. Inaddition, the known methods do not result in a particle size such thatthe D50 value is less than 10 μm without micronization. In addition,micronization would destroy the polymorphic purity of the product.

SUMMARY OF THE INVENTION

The present invention provides a scaleable and industrial productionprocess that results in a Nor-UDCA preparation or Bis-nor-UDCApreparation with desired quality attributes and pharmaceuticalapplicability. The inventors surprisingly found that purification andoptionally recrystallization of the potassium salt of Nor-UDCA withsubsequent precipitation of the free acid provides a novel physicallypure and thermodynamically stable crystalline form of Nor-UDCA (“FormA”).

In a first aspect, the present invention therefore relates to a purepolymorph of Nor-UDCA or Bis-nor-UDCA, or of a pharmaceuticallyacceptable salt thereof. The polymorph is thermodynamically stable.

Preferably, the Nor-UDCA, Bis-nor-UDCA, or pharmaceutically acceptablesalt thereof is in its anhydrous form. That is, the polymorph crystalscontain substantially no water. The amount of water in the crystals isgenerally less than 1%, preferably less than 0.5%, more preferably lessthen 0.1%, based on the total weight of the crystal.

The polymorph is characterized by XRPD peaks at 11.9, 14.4, 15.3, 15.8,and 16.6±0.2 degrees of 2-theta. Preferably, the polymorph ischaracterized by the XRPD pattern as shown in FIG. 4 for “Form A”.

A second aspect of this invention is a pharmaceutical compositioncomprising the polymorph described herein. The pharmaceuticalcomposition preferably exhibits a specific particle size distributionwherein at least 60% of the particles have a size <10 μm.

A third aspect of the invention is the use of the polymorph of thepresent invention or of the pharmaceutical composition of the presentinvention, for the treatment of cholestatic liver disease. Preferably,the cholestatic liver disease is selected from the group consisting ofprimary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC),autoimmune hepatitis (AIH) and overlap syndromes, including AIH-overlapsyndromes.

The polymorph or the pharmaceutical composition described herein mayalso be used for the treatment of metabolic liver disease. The metabolicliver disease may be non-alcoholic steato-hepatitis or alcoholicsteato-hepatitis.

The pharmaceutical composition of the present invention may beformulated for oral, parenteral, subcutaneous, intravenous,intramuscular, nasal, inhalative, topical or rectal administration. Itwill usually comprise one or more pharmaceutically acceptableexcipients.

A fourth aspect of the present invention is a method for the preparationof a pure polymorph of Nor-UDCA or Bis-nor-UDCA, or of apharmaceutically acceptable salt thereof, comprising the followingsteps: crystallizing the potassium salt of Nor-UDCA or Bis-nor-UDCA; andoptionally dissolving the potassium salt in a solvent and acidifying thesolution to obtain pure Nor-UDCA or Bis-nor-UDCA.

The solvent in which the potassium salt is dissolved is preferably amixture of water and acetone; and the precipitation is carried out byacidifying the solution to have a pH in the range of 1 to 2.

The process described herein leads to the formation of a single solidform of Nor-UDCA (or Bis-nor-UDCA), the crystal structure of which couldbe refined as monoclinic C2, closed packed without any solventaccessible void. The described process does not show the formation ofpolymorphic Nor-UDCA or Bis-nor-UDCA. Conversion into other polymorphicforms could also not be observed. The crystalline structure of Nor-UDCAobtained from the synthetic route proves to be the thermodynamicallystable form.

Further, the conditions of precipitation of Nor-UDCA from its potassiumsalt are as such that crystals of a desired particle size can beobtained directly in one step of the production process. An additionalmilling step to control the particle size of the crystals is thereforenot required. This is of great advantage taking into consideration thathigh-energy operations like grinding and milling (micronization) ingeneral lead to amorphization of Nor-UDCA, thereby to reducedpolymorphic purity end chemical purity.

In addition, the yield of Nor-UDCA with pharmaceutical pure quality bythis process is at least 45% of the source material and therefore veryhigh compared to published methods.

In summary, it was surprisingly found that the described method ofproduction for Nor-UDCA leads to a single-polymorphic, pure andcrystalline substance and does not need micronization, as it is forinstance established during the production of UDCA, the preparationcurrently in use for the treatment of cholestatic liver diseases. Thedescribed process is applicable to the preparation of Bis-nor-UDCAaccordingly.

The present invention is defined in the claims. The invention furtherrelates to the following aspects (1) to (17):

(1) A pure polymorph of Nor-UDCA or Bis-nor-UDCA, or of a pharmaceuticalacceptable salt thereof.

(2) The polymorph of item (1), which is thermodynamically stable.

(3) The polymorph of item (1) or (2), wherein said Nor-UDCA,Bis-nor-UDCA, or pharmaceutically acceptable salt thereof is in itsanhydrous form.

(4) The polymorph of any one of items (1) to (3), characterized by XRPDpeaks at 11.9, 14.4, 15.3, 15.8, and 16.8±0.2 degrees of 2-theta.

(5) The polymorph of item (4), characterized by the XRPD pattern asshown in FIG. 5.

(8) A pharmaceutical composition comprising the polymorph according toany one of items (1) to (5).

(7) The pharmaceutical composition according to item (6), wherein theparticle size distribution in the pharmaceutical composition comprisesat least 80% particles with a size <10 μm.

(8) The polymorph according to any one of items (1) to (5), or-thepharmaceutical composition according to item (6) or (7), for thetreatment of cholestatic liver disease.

(9) The polymorph or the pharmaceutical composition according to item(8), wherein the cholestatic liver disease is selected from the groupconsisting of primary biliary cirrhosis (PBC), primary sclerosingcholangitis (PSC), autoimmune hepatitis (AIH) and overlap syndromes,including AIH-overlap syndromes.

(10) The polymorph according to any one of items (1) to (5), or thepharmaceutical composition according to item (8) or (7), for thetreatment of metabolic liver disease and/or arteriosclerosis.

(11) The polymorph or the pharmaceutical composition according to item(10), wherein the metabolic liver disease is non-alcoholicsteato-hepatitis.

(12) The polymorph or the pharmaceutical composition according to item(10), wherein the metabolic liver disease is alcoholic steato-hepatitis.

(13) The pharmaceutical composition according to any one of items (8) to(12), which is formulated for oral, parenteral, subcutaneous,intravenous. Intramuscular, nasal, topical or rectal administration.

(14) The pharmaceutical composition according to any one of items (8) to(13), comprising one or more pharmaceutically acceptable excipients.

(15) A method for the preparation of a pure polymorph of Nor-UDCA orBis-nor-UDCA, or of a pharmaceutically acceptable salt thereof,comprising the following steps:

-   -   crystallizing the potassium salt of Nor-UDCA or Bis-nor-UDCA;        and    -   optionally dissolving the potassium salt in a solvent and        acidifying the solution to obtain pure UDCA or Bis-nor-UDCA.

(16) The method of item (15), wherein said solvent is a mixture of waterand acetone, and wherein said precipitation is carried out by acidifyingthe solution to have a pH in the range of 1 to 2.

(17) The method of item (15) or (16), further comprising the followingsteps: converting a compound of formula (A)

into a compound of formula (B)

(b) converting the compound of formula (B) into a compound of formula(C)

(c) converting the compound of formula (C) into a compound of formula(D) in crude form

and(d) treating the compound of formula (D) in crude form with KOH underconditions to crystallize the potassium salt of Nor-UDCA orBis-nor-ODCA;wherein n is 0 or 1.

DETAILED DESCRIPTION OF THE INVENTION Polymorph

The present invention provides a pure polymorph of Nor-UDCA orBis-nor-UDCA, or of a pharmaceutically acceptable salt thereof. Thepolymorph is thermodynamically stable.

Polymorphism is defined as the ability of a substance to crystalline inmore than one crystal lattice arrangement. Polymorphism can influencemany aspects of solid state properties of a drug. Different crystalmodifications of a substance may differ considerably from one another inmany respects such as their solubility, dissolution rate and finallybioavailability. An exhaustive treatment of polymorphism inpharmaceutical and molecular crystals is given e.g. by Byrn (Byrn, S.R., Pfeiffer, R. R., Stowell, J. G., “Solid-State Chemistry of Drugs”,SSCI Inc., West Lafayette, Ind., 1999), Brittain, H. G., “Polymorphismin Pharmaceutical Solids”, Marcel Dekker, Inc., New York, Basel, 1999)or Bernstein (Bernstein, J., “Polymorphism in Molecular Crystals”,Oxford University Press, 2002).

The term crystalline refers to any non-amorphous form of the activepharmaceutical ingredient (API). The term “amorphous form” refers to aform of the API which has no long-range order like crystallinestructures. The atoms or molecules of a material present in amorphousform are arranged in a non-uniform array. It is for example possible todistinguish amorphous from crystalline forms of a compound by powderX-ray diffraction.

The term “crystalline polymorph” or “polymorph” as described herein,refers to a specific crystal form of an active pharmaceutical ingredientwhich can be characterized by analytical methods such as e.g. X-raypowder diffraction or IR-spectroscopy.

Preferably, the Nor-UDCA, Bis-nor-UDCA, or pharmaceutically acceptablesalt thereof is in its anhydrous form. That is, the polymorph crystalscontain substantially no water. The amount of water in the crystals isgenerally less than 1%, preferably less than 0.5%, more preferably lessthan 0.1 %, based on the total weight of the crystal.

A polymorph is “pure” in the sense of the present invention if it issuitable for pharmaceutical application and contains less than 2%impurities. The amount of impurities in the polymorph of the presentinvention is generally less than 2%. preferably less than 1%, morepreferably less than 0.5%, more preferably less than 0.1% based on thetotal weight of the preparation. The amount of any single impurity inthe polymorph of the present invention is preferably less than 0.1 %,more preferably less than 0.05%, most preferably less than 0.03% basedon the total weight of the preparation. In a first embodiment, the totalamount of impurities in the polymorph of Nor-UDCA is less than 2%,preferably less than 1%, more preferably less than 0.5%, more preferablyless than 0.1% based on the total weight of the Nor-UDCA. The amount ofany single impurity in the polymorph of Nor-UDCA is preferably less than0.1 %, more preferably less than 0.05%, most preferably less than 0.03%based on the total weight of the Nor-UDCA. In a second embodiment, thetotal amount of impurities in the polymorph of Bis-nor-UDCA is less than2%, preferably less than 1%, more preferably less than 0.5%. morepreferably less than 0.1% based on the total weight of the Bis-nor-UDCA.The amount of any single impurity in the polymorph of Bis-nor-UDCA ispreferably less than 0.1 %. more preferably less than 0.05%, mostpreferably less than 0.03% based on the total weight of theBis-nor-UDCA.

Impurities can be determined as described herein in Example 2, Theimpurity profile of Nor-UDCA is specified by known and unknownimpurities. Known impurities are Ursodeoxycholic acid (UDCA) and3α,7βdihydroxy-24-nor-5β-cholane-23-amide (Amide). UDCA, also referredto as impurity A, is the starting material of the synthesis of Nor-UDCAwhile the Amide, also referred to as impurity B, represents anintermediate formed in step 3 of the synthesis of Nor-UDCA. Unknownimpurities might result from the synthetic route hut also from thedegradation of Nor-UDCA.

The polymorph of the present invention is preferably single-polymorphic,i.e. it essentially consists of a single polymorph, and/or it haspolymorphic purity. The amount of amorphous Nor-UDCA (or Bis-nor-UDCA,respectively) in the polymorph of the present invention is typicallynegligible. Preferably, no amorphous Nor-UDCA or Bis-nor-UDCA in thepolymorph of the present invention is detectable, e.g. by XRPD. Morepreferably, the polymorph of the invention contains substantially noamorphous Nor-UDCA (or Bis-nor-UDCA, respectively). Most preferably thepolymorph of the invention does not contain any amorphous Nor-UDCA (orBis-nor-UDCA, respectively). The amount of the polymorph of Form A inthe polymorph of the present invention is preferably at least 90%, morepreferably at least 90.5%, more preferably at least 99.9%, mostpreferably substantially 100%, based on the total weight of the Nor-UDCA(or Bis-nor-UDCA, respectively).

The polymorph of the invention is thermodynamically stable. Thepolymorph of the invention was found with high occurrence in all typesof crystallisation modes and also formed from different pure solventsand mixtures. Even in the crystallization experiments starting withamorphous form of NorUDCA produced by grinding (for vapour diffusiononto solids) or by evaporation of freeze-dried solution(cooling/evaporation experiments) to erase memory effects of Form A, theXRPD analyses performed on the isolated solids showed that predominantlyForm A was obtained. Surprisingly, the invention allows to grow singlecrystals of Form A.

The particle size distribution of the polymorph of the invention ispreferably such that at least 60% of the crystals have a particle sizeof less than 10 μm.

The polymorph of the present invention preferably has a OS0 of less than10 μm. For example, the D50 may range from 0.5 μm to 10 μm, morepreferably from 1 μm to 9 μm, more preferably from 2 μm to 8 μm, mostpreferably from 3 μm to 7 μm. The polymorph of the present inventionpreferably has a D90 of less than 30 μm. For example, the D90 may rangefrom 2 μm to 30 μm, more preferably from 5 μm to 25 μm, more preferablyfrom 8 μm to 20 μm, most preferably from 10 μm to 18 μm. The polymorphof the present invention preferably has a D95 of less than 30 μm. Forexample, the D95 may range from 3 μm to 30 μm, more preferably from 6 μmto 28 μm, more preferably from 9 μm to 25 μm, most preferably from 10 μmto 20 μm.

D50, D90 and D95 represent the median or the 50th percentile, the90^(th) percentile and the 85th percentile of the particle sizedistributions respectively, as measured by volume. That is, D50 (D90;D95) is a value on the distribution such that 50% (90%; 85%) of theparticles have a volume of this value or less.

The particle size distribution can be determined as described herein inExample 3 and/or according the European Pharmacopeia (Ph. Eur.), edition6.8. section 2.9.31, preferably with a Mastersizer 2000 by Malverninstruments. The evaluation is typically carried out by the Fraunhofermodel.

Method for Preparing the Polymorph

The method for preparing the polymorph of the invention preferablycomprises the following steps:

(a) converting a compound of formula (A)

into a compound of formula (B)

(b) converting the compound of formula (B) into a compound of formula(C)

(c) converting the compound of formula (C) into a compound of formula(D) in crude form

and

(d) treating the compound of formula (D) in crude form with KOH underconditions to crystallize the potassium salt of Nor-UDCA orBis-nor-UDCA:

wherein n is 0 or 1.

The potassium salt obtained in step (d) can be converted into the pureform of compound (D) by dissolving the potassium salt in a solvent,acidifying the solution so as to obtain crystals of pure compound (D).

When n=1, the starting compound of formula (A) is UDCA, and the productof formula (D) is Nor-UDCA.

When n=0, the starting compound of formula (A) is Nor-UDCA, and theproduct of formula (D) is Bis-nor-UDCA.

The solvent is preferably a mixture of 2-propanol and water, wherein the2-propanol may be added first, followed by the water until the potassiumsalt has dissolved completely.

In the following, preferred embodiments of the method of the invention,highlighting advantages thereof, are described. Each of the followingsteps, and each of the substeps thereof, can be combined with otherembodiments of this invention. In particular, any of the features of thefollowing embodiments can be combined with the embodiments describedabove.

Step 1: Preparation of 3α,7β-diformyloxy-5β-cholan-24 oic acid (I)

a) Process Description (Protection)

UDCA is added to formic acid and toluene (>3 hours at 65 to 75° C;reducing the temperature to 18 to 22° C.). The toluene phase isseparated and the formic acid/water phase is discharged. The toluenephase is concentrated (≦65° C.) and n-heptane is added quickly at 55 to65° C. to crystallize the product. The reaction mixture is cooled to 10to 15° C. and stirred. The suspension is filtered, washed with n-heptaneand dried at maximum 50° C. (LCD: ≦1.0%), Product I is obtained as awhite solid.

b) Differences Compared to the Published Synthesis

Perchloric acid was left out to simplify the process and addition ofacetic add anhydride was left out to avoid the powerful gas evolving. Toobtain a more pore product and a better crystallization process, thereaction mixture is in situ extracted into toluene and the product wascrystallized from toluene/n-heptane. Moreover, commercial aqueous formicacid could be employed and the need for anhydrous reaction conditionscould be avoided. The precipitation from toluene/heptane furthermoreserves as a method of further purifying the product. This step resultsin addition in an enhanced yield of approximately 85%.

Step 2: Preparation of 3α,7β-dihydroxy-24-nor-5β-cholan-23-nitrile (II)

a) Process Description (Re-Arrangement)

Trifluoroacetic acid, product I, and trifluoracetic acid anhydride aremixed (>40 min at 15 to 25° C.). The reaction mixture is stirred andcooled, while sodium nitrite is added (1.5 to 3 hours at 15 to 25° C.).The reaction mixture is very gently heated to 35 to 40° C. and thetemperature is kept constant for ≧45 min. Then, the temperature israised to 44 to 48° C. and kept constant for 30 min. After cooling to<22° C. toluene and water are added. The phases are separated and thewater/acid phase is discharged. Water is added to the toluene phase anddischarged after separation. Toluene is distilled off at <50° C. to givea highly viscous oil. EtOH, n-heptane and NaOH 28% are added to the oil,heated to 55-65° C. for 1.5 hrs, and product II starts to crystallize.Water is added and the suspension is cooled to 16 to 22° C. Thesuspension is filtered, the cake is washed with EtOH/water followed byn-heptane and dried <50° C. (LOD: ≦1.0%). Product II is obtained as awhite to light yellow solid.

b) Differences Compared to the Published Synthesis

The volume of trifluoroacetic acid was reduced to minimize the use ofharmful and expensive chemicals. To optimize safety and purity,temperature intervals and time intervals during addition of sodiumnitrite and heating were modified. The work-up procedure was modified inorder to be practical in production scale and to increase the purity Thereaction mixture was extracted with toluene and was afterwards fullydeprotected with sodium hydroxide in EtOH/water/n-heptane. The productwas crystallized from alkaline EtOH/water/n-heptane. This is in contrastto the published method by Schteingart (1988), a method withoutcrystallization of the product and thus no element of purification.

Yields obtained by the revised process are around 80%.

Step 3: Preparation of 3α,7β-dihydroxy-24-nor-5β-cholan-23oic acid (III)

a) Process Description (Hydrolysis)

Product II, n-propanol and sodium hydroxide (pellets) are mixed in asteel reactor. The reaction mixtured is stirred and refluxed untilhydrolysis is complete (>20 h). Water is added and n-propanol/water aredistilled off, keeping the temperature above 45° C. Toluene-Water isadded, while the reaction mixture is stirred efficiently and thetemperature is kept at 65 to 65° C. The pH is adjusted to 1.0 to 2.0with HCl 30%. Product III (crude nor-UDCA including impurities at ayield of app. 95 % by UV detection at 290 nm) crystallizes from thetwo-phase system during the pH adjustment. The suspension is cooled to18 to 22° C. The suspension is filtered and the filter cake is washedwith water and n-heptane and dried at maximum 50° C. (LOD: ≦1.0%).

b) Differences Compared to the Published Synthesis

The reaction time was significantly reduced by changing the solvent fromEtOH:water 1:1 to pure n-propanol and by changing from potassiumhydroxide to sodium hydroxide. The volume of solvent was also reduced.Washing and extraction were left out in the work-up procedure andinstead the product was crystallized directly from a two-phase system ofwater and toluene. This method is much more simplified: it safes time,reduces toxic solvents, uses non-toxic materials and thuscrystallisation is subject to improved control. Crystallisation fromthis two-phase system provided an excellent product quality With a yieldof at least 90%.

Step 4: Preparation of 3α,7β-dihydroxy-24-nor-5β-cholan-23-oic acidpotassium salt (Nor-UDCA potassium salt, IV)

a) Process Description (Purification)

Product III, 2-propanol and one equivalent of potassium hydroxide aremixed and heated to 70 to 80° C. Water is slowly added until a solutionis obtained. The solution is light yellow to yellow and slightly turbid.Active carbon and perlite are added and the solution is filtered whilehot. Under reduced pressure 2-propanol/water are distilled off at 40 to80° C. By addition of 2-propanolol, the distillation is continued untilthe water content is ≦2%. Thereafter, the suspension is cooled to 5 to15° C. (≧4 hours). The temperature is towered to 0 to 5° C. and productIV is filtered off, washed with 2-propanol and dried under reducedpressure ≦50° C. Product IV is obtained as a white solid. In case theobtained potassium salt is not of sufficient purity it can bere-crystalised: Product IV and 2-propanol are mixed and heated to 75 to80° C. Water is added until a solution is obtained, 2-Propanol/water aredistilled off under reduced pressure at 40 to 80° C. During thedistillation product IV starts crystallizing. Again, the distillation iscontinued until the water content is ≦2%. 2-Propanol is added and thesuspension is cooled to 5 to 15° C. (≧4 hours). The temperature islowered to 0 to 5° C. and product V is filtered off, washed with2-propanol and dried under reduced pressure ≦50° C. There-crystallization is repeated until the test for purity is compliant(product V is obtained pharmaceutical pure and at very high yields of≧98%, a proof of very efficient purification).

b) Differences Compared to the Published Synthesis

A new purification step of the synthesis is described, not published inthe state of the art.

Step 5: Preparation of Nor-UDCA Pure (VI)

a) Process Description (Final Precipitation)

Product V, water and acetone are stirred at 22 to 28° C. to obtain asolution. The solution is filtered. The pH of the reaction mixture isadjusted to 1.0 to 2.0 with slow addition of HCl 30%. Product VIimmediately starts crystallizing. The suspension is cooled to 18 to 24°C., filtered off, washed with water for injections, water forinjections/acetone, and n-heptane and dried under reduced pressure at≦50° C. (LOD: ≦0.8%). The yield is at least 90%.

b) Differences Compared to the Published Synthesis

In the published procedure Nor-UDCA is purified by ion exchange columnchromatography and re-crystallization from methanol/acetone. The mosteffective purification method was identified to be the formation and/oralternatively recrystallization of Nor-UDCA potassium salt from2-propanol/water followed by precipitation of the free acid fromwater/acetone. This purification method proved to be very effective inremoving both the known impurity A (amide) and B (UDCA) as well asunknown impurities. Finally, after sieving nor-UDCA is synthesized athigh quality with a defined particle size distribution (60% <10 μm) asrequired for pharmaceutical applications.

Pharmaceutical Compositions

The invention also relates to a pharmaceutical composition comprisingthe polymorph of the invention.

The pharmaceutical composition may comprise one or more suitableexcipients which are pharmaceutically acceptable.

According to a special embodiment of the present invention the polymorphcan be formulated for oral or intravenous administration, wherein theseformulations further comprise pharmaceutically acceptable carriers,adjuvants, excipients and/or vehicles.

Solid dosage forms for oral administration can include tablets,preferably effervescent or chewable tablets, capsules, pills, powdersand granules. In such solid dosage forms, the polymorph can be admixedwith regularly used substances like sucrose, mannitol, sorbitol, starchand starch derivatives, cellulose and cellulose derivates (e.g.microcrystalline cellulose), di-calcium phosphate, lactose, colloidalanhydrous silica, talc, lubricating agents (e.g. magnesium stearate,macrogols), disintegrants and buffering agents. Tablets and pills canalso be prepared with enteric coatings in order to prevent that API isaffected by the stomach acids and enzymes.

Liquid dosage forms for oral administration can include pharmaceuticallyacceptable emulsions, solutions, suspensions and syrups containing inertdiluents commonly used in the art, such as water or ethanol/watermixtures. These dosage forms may contain microcrystalline cellulose,alginic acid or sodium alginate, methylcellulose and alike to adjustrheological properties, sweeteners/flavouring agents, and/or employingsorbic acid or other suitable antimicrobial preservatives. Whenadministered by nasal aerosol or inhalation, the compositions accordingto the present invention may be prepared as solutions in saline,employing benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons and/or othersolubilizing or dispersing agents.

Suppositories for rectal administration of the API can be prepared bymixing the polymorph with a suitable non-irritating excipient such ashard fat, cocoa butter and polyethylene glycols which are solid at roomtemperature but liquid at rectal temperature, such that they will meltin the rectum and release the API and optionally other active compoundspresent in said suppositories.

Injectable preparations, for example sterile injectable aqueous oroleaginous suspensions, can be formulated according to the known artusing suitable dispersing agents, wetting agents and/or suspendingagents. The sterile injectable preparation can also be a sterileinjectable solution or suspension in a nontoxic parenterally acceptablediluent or solvent. Among the acceptable vehicles and solvents that canbe used are water and isotonic sodium chloride solution. Sterile fixedoils are else conventionally used as a solvent or suspending medium.

The dosage forms comprising the polymorph of the invention can furtherinclude conventional excipients, preferably pharmaceutically acceptableorganic or inorganic carrier substances which do not react with theactive compound. Suitable pharmaceutically acceptable carriers include,for instance, water, salt solutions, alcohol, oils, preferably vegetableoils, polyethylene glycols, gelatin, lactose, amylose, magnesiumstearate, surfactants, perfume oil, fatty acid mono-glycerides anddiglycerides, petroethral fatty acid esters, hy-droxymethyl-cellulose,polyvinylpyrrolidone and the like. The pharmaceutical preparations canbe sterilized and if desired, mixed with auxiliary agents, likelubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringand/or aromatic substances and the like which do not deleteriously reactwith the active compounds. For parenteral application, particularlysuitable vehicles consist of solutions, preferably oily or aqueoussolutions, as well as suspensions, emulsions, or implants.

Various delivery systems are known and can be used to administer theAPI, including, for example, encapsulation in liposomes, emulsions,microparticles, microcapsules and microgranules (see, e.g., EP 1 317925). The required dosage can be administered as a single unit or in asustained release form. The required dosage form may further beadministered in a multiple unit form, in immediate, sustained,prolonged, or extended release form, prepared by coating, as a matrixformulation and the like.

The bioavailability of the API may be enhanced by micronization of theformulations using conventional techniques such as grinding, milling endspray drying in the presence of suitable excipients or agents such asphospholipids or surfactants. However, in a special embodiment nogrinding and milling is required as the polymorph of the inventionalready has a suitable particle size.

The API may be formulated in a pharmaceutically acceptable salt form.Pharmaceutically acceptable salts of the API include preferably metalsalts, in particular alkali metal salts, or other pharmaceuticallyacceptable salts. Pharmaceutically acceptable base addition saltsinclude metallic salts made from lithium, aluminum, calcium, magnesium,potassium, sodium and zinc or organic salts made from primary, secondaryand tertiary amines and cyclic amines.

The pharmaceutical composition comprises preferably an effective amountof Nor-UDCA or Bis-nor-UDCA and a pharmaceutically acceptable carrierand/or excipient.

According to a preferred embodiment of the present invention thepharmaceutical composition comprises 10 to 8000 mg, preferably 25 to5000 mg. more preferably 50 to 1500 mg, in particular 250-500 mg, ofNor-UDCA or Bis-nor-UDCA.

On average the Nor-UDCA, Bis-nor-UDCA and/or pharmaceutical acceptablesalts thereof may preferably be administered to a patient in an amountof 25 mg to 5 g, preferably 100 mg to 2.5 g, in particular 80.0 mg to1.5 g per day. However, 1 g of Nor-UDCA, Bis-nor-UDCA and/orpharmaceutical acceptable salts and esters thereof is most preferablyadministered to a patient. It is further noted that Nor-UDCA,Bis-nor-UDCA and/or pharmaceutical acceptable salts thereof may beadministered to an individual in 1-3000 mg/d, preferably 10-2000 mg/d,more preferably 100-1500 mg/d, e.g., 100, 200, 300, 400, 500, 600, 700,750, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1600 mg/d, in particular500 or 750 or 1000 or 1500 mg/d. Said amounts are administeredpreferably at once or possibly in more than one dose (at least 2, 3, 4,5 or 10 doses) per day. The drug or the pharmaceutical compositionaccording to the present invention may be administered for more than oneweek, preferably more than four weeks, more preferably more than sixmonths, most preferably more than one year, in particular lifelong.

Nor-UDCA, Bis-nor-UDCA or salts thereof can be administered not only incombination with pharmaceutically acceptable carriers and in dosageforms as described herein, but, of course, also in combination with oneor more additional active ingredients (e.g. ursodeoxycholic acid, NSAID,like sulindac and ibuprofen) which are also known to be effectiveagainst the same or a similar disease to be treated (e.g.ursodeoxycholic acid) or against another disease, which may bepreferably a result of a liver disease.

Pharmaceutical Use of the Polymorph

The liver disease to be treated according to the present invention maybe a cholestatic liver disease, preferably primary sclerosingcholangitis (PSC), primary biliary cirrhosis (PBC) or progressivefamilial intrahepatic cholestasis, in particular progressive familialintrahepatic cholestasis type 1, 2 and 3, cystic fibrosis, drug-inducedcholestasis or a noncholestatic liver disease such as chronic viralhepatitis (B, C, D), alcoholic and non-alcoholic steatohepatitis,autoimmune hepatitis, hemochromatosis, Wilson disease andalpha-1-antitrypsin deficiency.

The API of the invention may be used alone or in combination with otheranti-inflammatory drugs, like NSAIDs (e.g. ibuprofen, sulindac) and/orin combination with ursodeoxycholic acid or 5-aminosalicylic acid.

According to another preferred embodiment of the present invention theliver disease is primary sclerosing cholangitis (PSC) and/or primarybiliary cirrhosis (PBC).

According to another preferred embodiment of the present invention thedisease to be treated is a metabolic disease, e.g.non-alcoholic-steato-hepatitis, diabetes, and/or hyperlipidemia.According to yet another preferred embodiment of the present inventionthe disease to be treated is arteriosclerosis.

EXAMPLES Reference Example

Various properties of Nor-UDCA synthesized according to Schteingart andHofmann (1988) Journal of Lipid Research Vol. 29(10), 1387-1385 wereinvestigated.

Chemcial Purity

The chemical impurity profile of the synthesized Nor-UDCA was evaluatedby HPLC/RI. The following results were obtained:

Impurity A 0.15% Impurity B ≦0.03%    (reporting threshold) Unknownimpurity 1 (RRT = 8.53) 0.51% Unknown impurity 2 (RRT = 0.84) 0.09%Total 0.75%

The total amount of impurities should be less than 0.05% if the compoundis to be used for pharmaceutical purposes. The Nor-UDCA synthesizedaccording to Schteingart and Hofmann failed to meet this requirement. Itwas tried to further purify the compound by repeatedre-crystallizations. However, it was not possible to reduce the totalamount of impurities to less than 0.05%.

Particle Size Distribution

The particle size of the crystals obtained was analyzed by microscopy.The median particle size was found to be 13.37 μm, i.e. 50% of thecrystals had a size of 13.37 μm or less. 60% of the crystals had aparticle size of 15.64 μm or less. 95% of the particles had a particlesize of 46.64 μm or less. Less than 30% of the particles had a particlesize of 10 μm or less. This particle size distribution is not suitablefor the intended pharmaceutical preparation. Milling/micronization isnot desired as this would result in a reduction of the polymorphicpurity and/or of the thermodynamic stability of the Nor-UDCA.

Example 1 Polymorphic Purity of the Synthesized Nor-UDCA

The polymorph screening study was carried out in two phases. In Phase 1,the synthesized Nor-UDCA, designated Form A, was characterised by XRPD,digital imaging, thermal analysis (DSC and TGMS). The purity of thematerial was checked by HPLC/RI. A summary of the analytical methods isprovided in Table 1. A qualitative solubility determination with 20solvents was carried out in order to fit the data for the selection ofthe crystallization solvents to be employed in the polymorph screen.Additionally, the solids recovered by evaporation of solutions used inthe solubility experiments were analyzed by XRPD and digital imaging toobtain information on the potential form formation of new forms ofNor-UDCA (see Table 2). Grinding tests were conducted in order tosupport the definition of the experimental space for thiscrystallization mode. Amorphous material to be used as starting materialwas intentionally produced by freeze-drying from 1,4-dioxane/water(95/5%) solution and by grinding (ball-milling) for “cooling/evaporationand vapour diffusion into solids experiments”. In Phase 2, 217experiments divided across different crystallization modes wereperformed. The applied crystallization modes were:

-   -   Combined cooling/evaporative crystallization experiments        starting with amorphous material    -   Crystallization with forward anti-solvent addition    -   Slurry experiments at two temperatures    -   Vapour diffusion into solutions    -   Vapour diffusion onto amorphous solids    -   Crystallization by grinding with 24 solvents and one dry.

Upon completion of the experiments, XRPD and digital images analyseswere performed on all crystallized solids. Following the identificationof stable forms, the assessment of their nature and relative stabilityversus the starting material form was proved by thermal analyses andXRPD re-analyses after storage.

The results of the polymorph screen of Nor-UDCA showed that Form A wasfound with high occurrence in all types of crystallization modes andalso formed from different pure solvents end mixtures (see FIG. 1), Evenin the crystallization experiments starting with the amorphous form ofNor-UDCA produced by grinding or by evaporation of freeze-dried solutionto erase memory effects of Form A, the XRPD analyses performed on theisolated solids showed that mainly Form A was obtained. It appears to beeasy to grow single crystals of Form A, as single crystals were observedto have grown in the vapour diffusion into solution experiments. Thatsuggests that gradually increasing the supersaturation by the slowdiffusion time of anti-solvent plays a role in allowing the crystalgrowth of Form A. The generally observed crystal habit was bulky-like.The crystal structure of Nor-UDCA was determined from single-crystalX-ray diffraction data collected at 294K and crystallized in themonoclinic system with C2 space group (Z′=2) (see Table 3). The crystalstructure of Nor-UDCA was successfully refined with a GOF of 1.002 (seeTable 3). The crystal packing and H-bonding scheme of Nor-UDCA ispresented in FIG. 2. Nor-UDCA presented only intramolecular H-bondsforming monomers repeating periodically. H-bonds are formed betweendifferent hydroxyl groups, Nor-UDCA molecules are closed packedfollowing a zigzag pattern and without any solvent accessible void (seeFIG. 3).

Three additional unstable forms (Form B, Form C1 and Form C2) wereidentified in cooling/evaporation experiments. Form C1 was also obtainedfrom an anti-solvent followed by a combined cooling/evaporationexperiment. The XRPD's of the three forms are similar, even highlysimilar in the case of Forms C1 and C2, which indicates the possibilityof being isomorphic types of structures. Form C1 was sufficiently stableto allow for determination of its solvated nature. Due to theirinstability, the nature of Form B and Form C2 could not be determined.Having the similarities of the XRPD patterns as well as the similaritiesof the molecular structure of the crystallization solvents from whichthe forms were crystallized out, one may think that Forms B and C2 aresolvated structures as well.

Three additional unstable mixtures (Form A plus1, Form A plus2 and FormA plus3) were identified. The mixture Form A plus1 is a mixture of FormA with another possible form obtained from cooling/evaporationexperiments. The mixture Form A plus2 is a mixture of Form A withanother possible form obtained from slurry experiments at lowtemperature. The mixture Form A plus3 is a mixture of Form A withanother possible form obtained after de-solvation or transformation ofForm C1 (obtained from cooling/evaporation experiments) during storageat ambient conditions. Due to their low presence in mixtures andinstability, the nature of the three potential forms present in themixture could not be determined.

A summary of the forms of Nor-UDCA found during the polymorph screeningstudy is presented in Table 4 und FIG. 4. The established conditions forthe synthesis of pure Nor-UDCA (purification and recrystallization ofthe potassium salt of Nor-UDCA with subsequent precipitation of the freeacid), however, only provides the physically pure and thermodynamicallystable crystalline substance, i.e. Form A.

TABLE 1 Analytical procedures used in the polymorph screen (a) X-raypowder diffraction Equipment: The plates were mounted on a Bruker GADDSdiffractometer equipped with a Hi-Star area detector. Calibration: TheXRPD platform was calibrated using Silver behenate for the longd-spacings end Corundum for the short d-spacings. Data Collection: Datacollection was carried out at room temperature using monochromaticCuK_(α) radiation in the 20 region between 1.5° and 41.5°. Thediffraction pattern was collected in two 20 ranges (1.5° ≦ 20 ≦ 21.5°for the first frame and 19.5° ≦ 20 ≦ 41.5° for the second) with anexposure time of 90 s for each frame. (b) Thermal analysis Differentialscanning Melting properties were obtained from DSC thermograms, recordedwith a calorimetry: heat flux DCS822e instrument (Mettler-Toledo GmbH,Switzerland). The DSC8225e was calibrated for temperature and enthalpywith a small piece of indium (m.p. = 156.6° C.; ΔHf = 28.45 Jg⁻¹). Thesamples were heated in the DSC from 25° C. to 300° C., at a heating rateof 10° C./min. Dry N₂ gas, at a flow rate of 50 ml/min was used to purgethe DSC equipment during measurement. Thermogravimetric Mass loss due tosolvent or water loss from the crystals was determined by analysis:TGA/SDTA. Monitoring the sample weight, during heating in a TGA/SDTA851e instrument (Mettler-Toledo GmbH, Switzerland), resulted in aweight versus temperature curve. The TGA/SDTA851e was calibrated fortemperature with indium and aluminium. The sample crucibles were heatedin the TGA from 25 to 300° C. at a heating rate of 10° C./min. Dry N₂gas was used for purging. (c) Thermogravimetric analysis/massspectroscopy A Thermostar GSD 301 T2 quadruple mass spectrometer(Pfeiffer Vacuum GmbH, Asslar) coupled with a quartz capillary to theTGA instrument was used to identify released gasses. The ultimatedetection limit is 10⁻¹⁴ mbar, and sensitivity for Ar is 200 A/mbar. Amultiple Ion Detection (MID) measurement was performed with achanneltron voltage of 950 V. (d) Digital imaging and optical microscopyDigital images were collected employing a Philips PCVC 840K CCD camera.Optical microscopy images were made using a Leica MZ9.5 stereomicroscopeequipped with a Leica DC 300 digital camera. (e) Dynamic vapour sorptionMoisture sorption isotherms were made using a DVS-1 system (SurfaceMeasurement Systems, London, UK). (f) HPLC/RI HPLC equipment: MerckAmazon LC-01 LaChrom Column: Waters XBridge C18 (250 × 4.6 mm; 5 μm), T= 40° C. Mobile phase: Isocratic mode (ACN:MeOH:5 mM phosphate buffer pH3 = 30:40:50 RI-Detecter Merck L-7490

TABLE 2 Solubility assessment results of Nor-UDCA Solvent Solubility(mg/ml) Form (by XRPD) Pentane <13 Form A Cyclohexane <14 Form AN-Methyl-2-pyrrolidone 404 Form A Tert-butylmethylether <13 Notdetermined 1,4-Dioxane 21 Form A 1,2-Dimethoxyethane <14 Form A2-Butanone <14 Form A Dimethylsulfoxide 207 Not determined Water <12Form A 1,2-Ethanediol <14 Not determined 2,2,2-Trifluoroethanol 21 FormA Chloroform <14 Form A Methanol 70 Form A Nitrobenzene <13 Form ADichlormethane <13 Form A Acetone <14 Form A Tetrhydrofurane 41 Form ANitromethane <13 Not determined Toluene <12 Form A Acetonitrile <13 FormA

FIG. 1 shows the XRPD pattern of the starting material and the Form Aobtained from different crystallization experiments. Note: Form Astarting material in FIG. 1 means pure, polymorph, crystallinenor-ursodeoxycholic acid with defined particle size, used in thepolymorph screening studies described.

TABLE 3 Single crystal data and structure refinement for Nor-UDCA Form AIdentication code Form A Empirical formula C₂₃H₃₈O₄ Formula weight378.53 T[K]   297 (2) λ [Å] 0.71073 Crystal system Monoclinic Spacegroup C2 Unit cell dimensions a [Å] 23.246 (3) b [Å] 11.197 (2) c [Å]19.232 (4) β [°] 122.869 (13) V [Å³]  4204.4 (13) Z 8 D_(c) [g/cm³]1.196 μ [mm⁻¹] 0.080 F(000) 1664 Crystal size [mm³] 0.45 × 0.35 × 0.25 θrange for data collection 3 → 32.6 Reflections collected 23586Independent reflections 14414 [R_(int) = 0.0541] Completeness to θ =32.6 96.6% Max. and min. transmission 0.9804 and 0.9651Data/restraints/parameters 14414/1/505 Goodness-of-fit on F² 0.980 FinalR indices [l > 2σ(l)] R1 = 0.0679, wR2 = 0.1194 R indices (all data) R1= 0.1760, wR2 = 0.1513 Absolute structure parameter   0.2 (9)

FIG. 2 deplete the crystal packing and H-bond scheme for nor-UDCA FormA. Note: Box indicates the single crystal packing of Nor-UDCA (Form A).

FIG. 3 depicts the molecular structure and atom numbering scheme by twosymmetrically independent molecules of Nor-UDCA crystals.

TABLE 4 Summary of the forms of Nor-UDCA during the polymorph screeningstudy Form/ TGMS (%)/ DSC potential Stability solvent loss Theoret-endo- HPLC/ form and by Nature/ ical mass therms RI mixtures XRPDremarks loss (%) (° C.) purity Form A Form A Anhydrate — Endother-99.96% mic event melt 249.5 Form A Form A — — — — plus 1 Form A Form A —— — — plus 2 Form A Potential 26.44 Endother- — plus 2 solvate mic event26.52% CHCl₃ 93 1.14 molecules Exother- CHCl₃ mic event 105 Endother-mic event melt 251.3 Form B Form A — — — — Form C1 Form A — — — — FormC^(AS) Potential 11.24 Endother- solvate mic event 11.18% 91.8 tolueneEndother- 0.52 molecules mic event toluene melt 250.9 Form A — — — —plus3* Form C2 Form A — — — — Notes: ^(AS)Anti-solvent experimentfollowed by cooling/evaporation *Form C1 de-solvated and/or transformedduring storage at ambient conditions

FIG. 4 depicts the comparison of XRPD pattern of obtained new forms ofNor-UDCA and XRPD pattern of Form A. Note: Form A starting material inFIG. 4 means pure, polymorph, crystalline nor-ursodeoxycholic acid withdefined pedicle size. Explanations of the XRPD patterns shown in FIG. 4:

Besides Form A, three potential mixtures of Form A with other forms wereisolated. The presence of the new forms was indicated by the presence ofadditional peaks in the XRPD patterns of the solids, peaks that are notspecific to the XRPD of Form A. Due to the fact that the XRPD analysesshowed that the predominant form in the mixture is Form A, the potentialmixtures were designated: Form A plus1, Form A plus2 and Form A plus3.

The mixture designated Form A plus1 was found upon completion of acooling/evaporation experiment with anisole and methanol. Its XRPDpattern showed three additional peaks (next to the ones of Form A) atapprox. 8.77°, 14.70° and 20.51° (2theta). The XRPD re-analyses ofsolids stored at ambient conditions in well and experimental vial showedthat the mixture converted completely to Form A within 7 days.

The mixture designated Form A plus 2 was found upon completion of aslurry experiment at 5° C. with chloroform. The XRPD pattern of thesolid shows five additional peaks at approx. 6.41°, 12.75°, 12.89°,14.72° and 17.06° (2theta) in comparison with Form A. The XRPDre-analyses of solid stored at ambient conditions in well showed thatthe mixture converted completely to Form A within 11 days, but it wasstable in experimental vial (within the same time interval).

The designated Form A plus3 mixture was identified after the conversionof Form C1 (anti-solvent experiments with 2,2,2-trifluoroethanol andtoluene) stored at ambient conditions for 7 days. The XRPD pattern ofthe potential mixture Form A plus3 shows three additional peaks at6.74°, 715° and 10.25° (2theta) in comparison with Form A.

Form B was identified upon completion of the cooling/evaporationexperiment with 1,4-dioxane and tetrahydrofuran. The XRPD pattern ofForm B is similar to that of Forms C1 and C2 in the sense that the mainintense peaks common to Forms C1 and C2 are present in the XRPD of FormB as well. However, including Form B in the same isomorphic class offorms (as Form C1 and C2) was not straight forward possible due to thepresence of the preferred orientation effects. The XRPD re-analyses ofform B showed that it converted within 10 days to Form A upon storage inwell and experimental vials at ambient conditions.

Form C1 was found in the cooling/evaporation experiments with p-Xyleneand methanol. XRPD analyses of the solid stored at ambient conditions inthe measuring well showed that the form converted to Form A within 7days. Form C1 was also found in the anti-solvent experiments with2,2,2-trifluoroethanol and toluene. Also in this case, the formconverted to Form A within 7 days of storage in the well.

FIG. 5 separately shows the XRPD pattern of Form A. The polymorph ischaracterized by XRPD peaks at 11.9, 14.4, 15.3, 15.8, 18.8, 16.6±0.2°of 2-theta and having the general appearance as shown in FIG. 5.

Example 2 Chemical Purify of the Synthesized Nor-UDCA

The chemical impurity profile of the synthesized Nor-UDCA was evaluatedby HPLC/RI. The method including the instrumental conditions aresummarised in Table 5. The possible by-products from the synthesis areimpurity A, impurity B, impurity C, impurity D, impurity E and impurityF. Impurity A is the starting material ursodeoxycholic acid (UDCA).impurity b is 3α,7β-dihydroxy-24-nor-5β-cholane-23amide, an intermediateformed by incomplete nitrile hydrolysis. Impurity C isNor-Chenodeoxycholic acid (Nor-CDCA) which is a major impurity in thestarting material UDCA. Impurity D is cholic acid (CA), another majorimpurity in the starting material UDCA. Impurity E is theformyl-protected UDCA which does not undergo the desired reaction. It isonly a theoretically potential impurity as the formyl-protection groupswill be cleaved by the alkaline treatment which as a result gives againimpurity A. Impurity F is 3α,7β-dihydroxy-24-nor-5β-cholane-23-nitrile,another intermediate of the synthesis. The efficiency of the establishedpurification methods is demonstrated by the results of the HPLC purityanalyses of three batches of Nor-UDCA (see Table 6). Effective removalof the potentially known and unknown impurities by the recrystalizationof the potassium salt of Nor-UDCA and subsequent precipitation of thefree acid lead to chemically pure Nor-UDCA.

TABLE 5 Purity method including instrumental conditions HPLC/RI Column:Waters Symmetry C18 250 × 4.6 mm, 5 μm Mobile phase: 30 Acetonitrile 40Methanol 50 5 mM Phosphate buffer pH 3.0 Temperature: 40° C. Dectection:Refractive index detector Flow rate: 0.8 ml/min Injection volumn: 150 μl

TABLE 6 Results of purity determination of Nor-UDCA Batch Batch Batch40019721 40019902 40019898 Impurity A  ≦0.03%¹ ≦0.03% ≦0.03% Impurity B≦0.03% ≦0.03% ≦0.03% Any other impurity ≦0.03% ≦0.03% ≦0.03% Sum of allimpurities ≦0.03% ≦0.03% ≦0.03% ¹Representing the limit of quantitationof the analytical procedure

Example 3 Particle Size of the Synthesised Nor-UDCA

The precipitation of the free acid leads to Nor-UDCA particles of aconsistent and defined size distribution. Micronization by milling inorder to control the crystal size of Nor-UDCA can be avoided. Theresults of the particle size measurement of three batches of Nor-UDCAdemonstrate that the desired particle size of less than 10 μm can beobtained directly by the established conditions of precipitation (seeTable 7).

TABLE 7 Results of particle size determination of Nor-UDCA by laserdiffraction (Equipment: Malvern Laser Diffraction Analyzer; Method: Dry;System details: Lense range 300 mm, beam length 10.00 mm) Batch BatchBatch Particle size 40019721 40019902 40019898 1 μm < D(v, 0.5) < 8 μm6.4 μm 6.7 μm 5.4 μm

SUMMARY

Presented here, is the formation and re-crystallization of the Nor-UDCApotassium salt from a mixture of 2-propanol and water followed by aprecipitation of the free acid from a mixture of water and acetone. Thispurification method via the potassium salt proves to be very effectivein removing both known impurities as well as unknown impurities. Inconclusion, the process described is highly efficient to provide achemically pure, single-polymorphic, crystalline preparation of Nor-UDCAwith a defined particle size.

1. A chemically pure polymorph of Nor-UDCA or Bis-nor-UDCA, or of apharmaceutically acceptable salt thereof, wherein the total amount ofchemical impurities is less than 0.5%, and at least 60% of the polymorphparticles have a size <10 μm.
 2. The polymorph of claim 1, wherein thetotal amount of chemical impurities is less than 0.1%.
 3. The polymorphof claim 2, wherein the total amount of chemical impurities is less than0.05%.
 4. The polymorph of claim 1, which is thermodynamically stable.5. The polymorph of claim 1, wherein said Nor-UDCA, Bis-nor-UDCA, orpharmaceutically acceptable salt thereof is in its anhydrous form. 6.The polymorph of claim 1, characterized by XRPD peaks at 11.9, 14.4,15.3, 15.8, and 16.6±0.2 degrees of 2-theta.
 7. The polymorph of claim6, characterized by the XRPD pattern as shown in FIG.
 5. 8. Thepolymorph of claim 1, containing no amorphous Nor-UDCA or Bis-nor-UDCAin detectable amount.
 9. The polymorph of claim 1, having avolume-weighted average particle diameter D50 of less than 10 μm. 10.The polymorph of claim 1, having a volume-weighted average particlediameter D95 of less than 30 μm.
 11. A pharmaceutical compositioncomprising the polymorph according to claim
 1. 12. The pharmaceuticalcomposition according to claim 11, wherein the particle sizedistribution in the pharmaceutical composition comprises at least 60%particles with a size <10 μm.
 13. The pharmaceutical compositionaccording to claim 11, adapted for the treatment of cholestatic liverdisease.
 14. The pharmaceutical composition according to claim 13,wherein the cholestatic liver disease is selected from the groupconsisting of primary biliary cirrhosis (PBC), primary sclerosingcholangitis (PSC), autoimmune hepatitis (AIH) and overlap syndromes,including AIH-overlap syndromes.
 15. The pharmaceutical compositionaccording to claim 11, adapted for the treatment of metabolic liverdisease and/or arteriosclerosis.
 16. The polymorph or the pharmaceuticalcomposition according to claim 15, wherein the metabolic liver diseaseis non-alcoholic steato-hepatitis.
 17. The pharmaceutical compositionaccording to claim 15, wherein the metabolic liver disease is alcoholicsteato-hepatitis.
 18. The pharmaceutical composition according to claim11, which is formulated for oral, parenteral, subcutaneous, intravenous,intramuscular, nasal, topical or rectal administration.
 19. Thepharmaceutical composition according to claim 11, comprising one or morepharmaceutical acceptable excipients.
 20. A method for the preparationof a pure polymorph of Nor-UDCA or Bis-nor-UDCA, or of a pharmaceuticalacceptable salt thereof, comprising: crystallizing the potassium salt ofNor-UDCA or Bis-nor-UDCA; and optionally dissolving the potassium saltin a solvent and acidifying the solution to obtain pure Nor-UDCA orBis-nor-UDCA.
 21. The method of claim 20, wherein said solvent is amixture of water and acetone, and wherein said precipitation is carriedout by acidifying the solution to have a pH in the range of 1 to
 2. 22.The method of claim 20, further comprising: (a) converting a compound offormula (A)

into a compound of formula (B)

(b) converting the compound of formula (B) into a compound of formula(C)

(c) converting the compound of formula (C) into a compound of formula(D) in crude form

and (d) treating the compound of formula (D) in crude form with KOHunder conditions to crystallize the potassium salt of Nor-UDCA orBis-nor-UDCA; wherein n is 0 or
 1. 23. The method of claim 20,characterized in that it does not comprise a milling step and/or amicronization step.